Download Rumen microorganisms and fermentation

Arch Med Vet 46, 349-361 (2014)
REVIEW ARTICLE
Rumen microorganisms and fermentation
Microorganismos y fermentación ruminal
AR Castillo-Gonzáleza, ME Burrola-Barrazab*, J Domínguez-Viverosb, A Chávez-Martínezb
aPrograma
de Doctorado en Philosophia, Departamento de Nutrición Animal. Facultad de Zootecnia y Ecología, Universidad
Autónoma de Chihuahua, Chihuahua, México.
bFacultad de Zootecnia y Ecología, Universidad Autónoma de Chihuahua, Chihuahua, México.
RESUMEN
El rumen es un ecosistema complejo donde los nutrientes consumidos por los rumiantes son digeridos mediante un proceso de fermentación
realizado por los microorganismos ruminales (bacterias, protozoos y hongos). Dichos microorganismos están en simbiosis, debido a su capacidad de
adaptación e interacción, y mientras el rumiante proporciona el ambiente necesario para su establecimiento estos proporcionan energía al animal, la
que proviene de los productos finales de la fermentación. Dentro del rumen, los microorganismos coexisten en un entorno reducido y a un pH cercano
a la neutralidad. Estos microorganismos fermentan los sustratos presentes en la dieta del rumiante (azúcares, proteínas y lípidos). Sin embargo, el
proceso de fermentación no es 100% eficaz, ya que durante la fermentación existen pérdidas de energía, principalmente en forma de gas metano (CH4),
el que representa un problema medioambiental, ya que es un gas de efecto invernadero. Por consiguiente, para mejorar la eficiencia de los sistemas
de producción de rumiantes se han establecido estrategias nutricionales que tienen como objetivo manipular la fermentación ruminal mediante el uso
de aditivos en la dieta, como monensina, sebo, tampones, compuestos de nitrógeno, probióticos, etc. Estos aditivos permiten cambiar el proceso de
fermentación y mejorar la eficiencia animal, además disminuyen la pérdida de energía. El objetivo de este trabajo es revisar los procesos fermentativos
que tienen lugar en el rumen y aplicar los fundamentos de estos en el desarrollo de nuevas estrategias nutricionales que pudieran ayudar a mejorar los
procesos de digestión, de manera que se alcance una máxima producción.
Palabras clave: aditivos, microorganismos ruminales, simbiosis.
SUMMARY
The rumen consists of a complex ecosystem where nutrients consumed by ruminants are digested by fermentation process, which is executed by
diverse microorganisms such as bacteria, protozoa, and fungi. A symbiotic relationship is found among different groups of microorganisms due to the
diverse nature of these microbial species and their adaptability and interactions also coexist. The ruminant provides the necessary environment for the
establishment of such microorganisms, while the microorganisms obtain energy from the host animal from microbial fermentation end products. Within
the ruminal ecosystem, the microorganisms coexist in a reduced environment and pH remains close to neutral. Rumen microorganisms are involved in
the fermentation of substrates contained in thedietof the animals (carbohydrates, proteins and lipids). However, the fermentation process is not 100%
effective because there are energy losses mainly in the form of methane gas (CH4), which is a problem for the environment since it is a greenhouse gas.
In order to improve the efficiency of ruminant production systems, nutritional strategies that aim to manipulate ruminal fermentation using additives
in the diet such as monensin, tallow, buffers, nitrogen compounds, probiotics, and others have been used. These additives allow changing the ruminal
fermentation process in ways that produce better growth efficiency while decreasing energy loss. The purpose of this review is to contribute to a better
understanding of the fermentation processes taking place in the rumen, providing information that can be applied in the development of new nutritional
strategies for the improvement of the digestion process to achieve maximum production.
Key words: additives, ruminal microorganisms, symbiosis.
INTRODUCTION
The rumen is a complex ecosystem where nutrients
consumed by the microorganisms such as bacteria, protozoa, and fungi are digested anaerobically. The main end
products of fermentation are volatile fatty acids (VFAs) and
microbial biomass, which are used by the host ruminant. The
interaction between microorganisms and the host animal
results in a symbiotic relationship that allows ruminants to
Accepted: 20.03.2014.
* Perif. R. Almada km. 1, C.P. 31453 Chihuahua, Chih., México;
[email protected]
digest diets rich in fiber and low in protein. In the rumen
the environment favors the microorganisms to provide the
enzymes necessary to digest the nutrients. Ruminants have
the ability to convert the low quality fibrous materials into
products such as meat, milk and fibers, which are useful to
humans. The ability of ruminal microorganisms to produce
the enzymes necessary for fermentation processes allows
ruminants to efficiently obtain the energy contained in
forages (Burns 2008). However, the ruminal fermentation
process is not completely efficient because it produces
some final products such as methane gas (Kingston-Smith
et al 2012) and excess ammonia (Russell and Mantovani
2002). Ruminants such as cattle, sheep, and goats have
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CASTILLO-GONZÁLEZ ET AL
evolved to use fibrous food efficiently (Oltjen and Beckett
1996). The anatomical adaptation of their digestive system
allows them to use cellulose as an energy source without
requiring external sources of vitamin B complex (Russell
and Mantovani 2002) or essential amino acids because
ruminal microorganisms are able to produce such products
(Cole et al 1982). Thus, a symbiotic relationship exists
within the rumen providing the necessary environment
for the establishment of microorganisms and substrates
required for their maintenance. In turn, the microorganisms
provide nutrients to the host ruminant to generate energy
(Russell and Rychlik 2001). The continued increase of
the human population has increased the need for more
and better animal products. For this reason, in the field
of animal production, most efforts have been directed to
increase ruminant production using biotechnological tools
to manipulate the ruminal ecosystem. For example, the use
of additives in the diet such as monensin, tallow, buffers,
nitrogen compounds, probiotics, etc., allow manipulation
of the ruminal fermentation process to maximize the
production efficiency while decreasing energy loss, for
example, methane which pollutes the environment. The
objective of this review is to describe the fermentation
processes in the rumen so that current knowledge can be
applied in the development of nutritional strategies for
improving animal production.
PHYSICOCHEMICAL PROPERTIES OF THE
RUMEN
The ruminant digestive system is composed of reticulum, rumen, omasum, and abomasum. The rumen is
mainly where the major fermentation processes are held
(Tharwat et al 2012 ). Enzymes present in the rumen are
produced by microorganisms. These enzymes are used
to digest and ferment food eaten by ruminants, thus, the
rumen is considered as a fermentation vat (Aschenbach
et al 2011). The main factors influencing the growth and
activity of ruminal microbial populations are temperature, pH, buffering capacity, osmotic pressure and redox
potential. These factors are determined by environmental
conditions. The rumen temperature is maintained in the
range of 39 to 39.5 °C (Wahrmund et al 2012) and may
increase up to 41 °C immediately after the animal eats
because the fermentation process generates heat (Brod
et al 1982). The pH depends on the production of saliva,
the generation and absorption of short-chain fatty acids
(SCFA), the type and level of feed intake, and the exchange of bicarbonates and phosphates through the ruminal
epithelium (Aschenbach et al 2011). Thus, these factors
determine both pH and buffering capacity in the reticule
ruminal environment. The pH constantly changes (Russell
and Strobel 1989), but it usually remains in the range of
5.5 to 7.0 (Krause and Oetzel 2006), depending on the diet
and buffering capacity of saliva, because saliva production is a constant process that provides bicarbonates and
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phosphates into the rumen. Furthermore, reticuloruminal
secretions also possess buffering capabilities, so this
environment does not only depend of the buffering capacity of saliva, which has a pH of 8.2 (Krause and Oetzel
2006). Generally, the bacterial intracellular pH remains
near 7.0, which decreases considerably when the cell is
under acidic environment. Likewise, microbial enzymes
are sensitive to changes in pH, for example, inhibition of
bacterial growth under acidic pH. This may be due to the
imbalance of intracellular hydrogen ions (Russell and
Wilson 1996). The osmotic pressure in the rumen depends
on the presence of ions and molecules, which generate a
gas tension (Lodemann and Martens 2006). The ruminal
fluid osmolality is approximately 250 mOsm/kg. Ruminal
fermentation processes may depend on the environmental
conditions and the type of diet, so these factors may influence the osmotic pressure of the rumen. Immediately
after feed intake, the osmotic pressure increases from 350
to 400 mOsm and then decreases gradually over a period
of 8 to 10 hours. The osmotic pressure increases with the
presence of VFAs produced by fermentation processes
and has a direct relationship with the pH in diets rich in
carbohydrates (Lodemann and Martens 2006).
RUMINAL MICROORGANISMS
The ruminal ecosystem consists of a wide diversity of
microorganisms that are in a symbiotic relationship in a
strict anaerobic environment (Ozutsumi et al 2005). The
microbiota is formed by ruminal bacteria, protozoa, and
fungi, at concentrations of 1010, 106, and 104 cells/ml,
respectively. Bacterial populations are most vulnerable to
the physicochemical properties of the rumen (McAllister
et al 1990).
RUMINAL BACTERIA
The rumen contains a variety of bacterial genera (table 1),
which constitute the majority of the microorganisms that
live in anaerobic environment (Pitta et al 2010). The
competition between bacteria in the rumen is determined
by several factors, among which are the preference for
certain substrates, energy requirements for maintenance,
and resistance to certain metabolism products that can be
toxic (Russell et al 1979).
CELLULOSE-DEGRADING BACTERIA
The ruminant diet is based on plant-based feed consumption. Because cellulose is the main component of
the cell wall of these plants, cellulolytic ruminal microorganisms play an important role in animal nourishment
(Russell et al 2009). Cellulose is digested in the rumen
(Michalet-Doreau et al 2002). The ability to degrade
cellulose depends mainly on the type of forage, crop
maturity, and the members of the cellulolytic bacterial
ADDITIVES, RUMINAL MICROORGANISMS, SYMBIOSIS
Table 1. Characteristics of principal ruminal bacteria.
Características de las principales bacterias ruminales.
Microorganisms
Gram stain
Morphology
Fermetation products
Cellulose-degrading bacteria
Fibrobacter succinogenes
Butyrivibrio fibrisolvens
Negative
Negative
Bacillus
Bacillus curve
Ruminococci albus
Positive
Cocci
Clostridium lochheadii
Positive
Amylolytic bacteria
Bacteriodes ruminicola
Ruminobacter amylophilus
Selenomonas ruminantium
Succinomonas amylolítica
Streptococci bovis
Negative
Negative
Negative
Negative
Positive
Bacillus
Bacillus
Bacillus curve
Oval
Cocci
Lipolytic bacteria
Anaerovibrio lipolytica
Negative
Bacillus
Lactate-degrading bacteria
Selenomonas lactilytica
Megasphaera elsdenii
Negative
Positive
Pectin-degrading bacteria
Lachnospira multiparus
Positive
Bacillus curve
Acetate, Formate, Lactate, H2,
CO2
(Duskova and Marounek
2001)
Ruminal archaea (methanogens)
Methanobrevibacter ruminantium
Positive
Bacillus
CH4 (of H2+CO2 or Formate)
(Yanagita et al 2000,
Hook et al 2010)
Methanomicrobium mobile
Negative
Bacillus
CH4 (of H2+CO2 or Formate)
Lactic acid-utilizing bacteria
Megasphaera elsdenii
Negative
Cocci
Succinate, Acetate, Formate
Acetate, Formate, Lactate,
Butyrate, H2, CO2
Acetate, Formate, H2, CO2
Bacillus (espores) Acetate, Formate, Butyrate, H2,
CO2
Formate, Acetate, Succinate
Formate, Acetate, Succinate
Acetate, Propionate, Lactate
Acetate, Propionate, Succinate
Lactate
Acetate, Propionate, Acetate
Bacillus curvado Acetate, Succinate
Cocci
Acetate, Propionate, Butyrate,
Valerate, H2, CO2
communities (Fondevila and Dehority 1996). To ensure
maintenance and growth of cellulolytic bacteria, optimal
ruminal conditions are required. A neutral pH near neutrality between 6 and 9 is best, while a pH less than 5.5
affects fiber digestibility (Weimer 1996). A temperature
of 39 °C affects the adhesion ability of bacteria to feed
particles (Michalet-Doreau et al 2001), while the presence
of extracellular cellulase enzymes (Weimer 1996) to break
β-glycosidic bonds (1-4) of the biopolymer provides sugars
for use by microorganisms (Wedekind et al 1988). In
addition, the presence of ionized calcium (Ca+2) favors the
establishment of such bacteria, except for F. succinogenes
Lactate
Reference
(Ivan et al 2012)
(Weimer 1996)
(Michalet-Doreau et al
2002)
(Weimer 1996)
(Cotta 1988)
(Cotta 1992)
(Cotta 1988, McAllister
et al 1990)
(Fuentes et al 2009)
(Brown et al 2006)
(Counotte and Prins
1981)
(Morales and Dehority 2009). The establishment of this
bacterial group can be affected by the presence of certain
types of lipids in the diet. For example, medium-chain fatty
acids are often toxic to cellulolytic bacteria, reducing the
digestibility of the fiber. For amylolytic bacteria (table 1),
starch is an important component of the diet of cattle and
high milk-producing cows which are fed with concentrates
containing major proportions of grains. Although these
diets for ruminants have been effective as a fermentable
energy source, they are also associated with metabolic
disorders such as acidosis (Gressley et al 2011), low-fat
milk syndrome and liver abscesses (Owens et al 1998).
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CASTILLO-GONZÁLEZ ET AL
In ruminants fed mainly forage, this bacterial species is
found at a concentration of 104-107cells/grams. In addition,
when the fermentable sugar concentration increases, its
concentration may be greater than 1011/grams in ruminal
contents (Nagaraja and Titgemeyer 2007). Furthermore,
S. bovis ferments glucose to provide acetate, formate, and
ethanol as a final product. However, in high concentrate
diets, this species changes its metabolism to provide
lactic acid as the final product, which causes a drop of
pH to 5.5 that is detrimental to the ruminant (Russell
and Hino 1985). To avoid this situation, it is necessary
to gradually introduce fermentable carbohydrates in the
ruminant diet. This dietary management allows S. bovis
not to produce lactic acid rapidly. Thus, the growth of
other starch-degrading bacteria, such as Bacteroides
ruminicola, Ruminobacter amylophilus, Selenomonas
ruminantium and Succinomonas amylolytica which produce other VFAs such as formate, acetate, propionate,
and succinate, is also promoted so that an imbalance of
homeostasis in the biochemical pathways in the ruminal
environment is avoided (Cotta 1992).
LACTATE-DEGRADING BACTERIA AND
LACTATE- BACTERIA
They have a very important role in the rumen (table 1)
mainly in those ruminants that are fed with high grains in
the diet. These bacteria metabolize lactic acid and control
its accumulation, which helps to keep the pH in the proper
range (Mackie and Heath 1979). This type of bacteria
increases when the diet consists of approximately 70%
concentrate (Brown et al 2006).
PECTIN-DEGRADING BACTERIA
They are important because the pectin represents
10-20% of total carbohydrates in forages used in ruminant
nutrition (table 1). Pectin is fermented by both bacteria and
protozoa (Dehority 1969) and the main bacteria that perform this function are Butyrivibrio fibrisolvens, Prevotella
ruminicola, Bacteroides ruminicola and Lachnospira
multiparus. These ruminal bacteria produce and release
pectinolytic enzymes into the ruminal environment; pectin
lyases are the primary enzymes that hydrolyze the pectin
in oligogalacturonoides (Duskova and Marounek 2001).
RUMINAL ARCHAEA OR METHANOGENS
They depend on the growth, maintenance, and activity of
a diverse population of microorganisms (table 1). However,
microbial activity is the main source of greenhouse gases
in agriculture (Mosoni et al 2011), such as methane gas.
Methane is an end product of ruminal fermentation and
is considered as a loss of total energy consumed by ruminants, representing 6-10% of total energy (Mohammed
et al 2004), which contributes to the greenhouse effect
352
(Garnsworthy et al 2012). In ruminants, 80% of methane
is generated during fiber fermentation, mainly cellulose,
and 20% of methane is generated by the decomposition
of manure (Vergé et al 2007). These percentages can vary
depending upon the composition of ruminant diet (Rotz
et al 2010). Methanogenesis is a necessary process because it is a way to maintain low H+ concentrations in the
ruminal environment by reducing CO2 (Bodas et al 2012).
Oxidative processes, for example, glycolysis occur in the
rumen, reduced substrates are generated this process. For
example, cofactors such as NADH, which is re-oxidized
to NAD+ to complete sugar fermentation, are produced.
NAD+ is regenerated by electron transfer through electron
acceptors other than O2, such as CO2, sulfate, nitrate, or
fumarate. Electron transport is linked to ATP generation
as a result of an electron gradient generated across the
membrane where these cofactors are present (Moss et al
2000). The production of H+ is a thermodynamically unfavorable process controlled by the potential of the electron
carriers (Moss et al 2000). Although H+ is one of the main
end products of the fermentation of bacteria, protozoa, and
fungi, it does not accumulate in the rumen because it is
rapidly used by some microorganisms that are a part of the
ecosystem. In the rumen, there exists an interrelationship
among species producing and utilizing H+ that is called
“interspecies H+ transfer”. The production of methane in
the ruminal environment is a clear example of this process,
where there is an association between species that produce
and utilize H+ (Walker et al 2012). Methanogenesis is the
main sink of H+ removal (Moss et al 2000). Methane is
generated by methanogenic bacteria utilizing the carbon
dioxide and hydrogen (van Zijderveld et al 2011). When H+
is not used by the methanogens, NADH can be reoxidized
by a dehydrogenase to produce ethanol or lactate. This
process occurs rapidly in animals fed with high amounts
of fermentable sugars (Moss et al 2000).The methanogens
belong to the domain Archaea (Morgavi et al 2010) and
the phylum Euryarchaeota. Not only CO2 is used by the
methanogens to produce CH4, but these microorganisms
can also degrade substrates containing methyl (CH3-) or
acetyl (CH3OO-) groups, such as methanol and acetate
that act as electron acceptors (Liu and Whitman 2008).
PROTEOLYTIC BACTERIA
In the rumen, the forage proteins and structural polysaccharides are degraded by 50-70% by the action of
microorganisms. Ruminal proteolysis is carried out by
enzymatic production of ruminal microorganisms by
protein hydrolysis processes, degradation of peptides,
and amino acid deamination (Cotta and Hespell 1986).
The main bacterial species with proteolytic activity are
Bacteroides amylophilus, Bacteroides rutminicola, and
Butyrivibrio fibrisolvens (Cotta and Hespell 1986). This
activity has also been reported in Streptococcus bovis and
Prevotella albensis (Sales-Duval et al 2002).
ADDITIVES, RUMINAL MICROORGANISMS, SYMBIOSIS
LIPOLYTIC BACTERIA
Lipids are modified by microbial fermentation, and
the unsaturated fatty acids present are transformed into
saturated fatty acids in the rumen. Ruminal microorganisms transform lipids by two major pathways, lipolysis
and biohydrogenation. The major types of lipids in the
diet are triglycerides, phospholipids, and galactolipids
(Jenkins et al 2008). The lipids in the rumen are first
hydrolyzed by microbial lipases. These lipases break the
ester bonds, releasing fatty acids (Liu et al 2009). After
lipolysis, unsaturated fatty acids are biohydrogenated due
to the presence of H+ produced by ruminal microorganisms
(Jenkins et al 2008). The rate of lipolysis depends mainly
on the type of lipids present in the diet (Beam et al 2000)
and the ruminal pH. A pH value less than 6.0 causes slow
lipolysis, which decreases as the pH drops (Fuentes et al
2009), therefore, lipolysis depends on the type of fermentable substrates in the diet (van Nevel and Demeyer
1996). A. lipolytica produces two hydrolytic enzymes, one
of which is bound to the cell membrane and the other is
extracellular (Henderson and Hodgkiss 1973), the activity
of these bacteria decreases in high concentrate diets, due
to the drop in pH (Loor et al 2004), the main function of
B. fibrisolvens is to biohydrogenate polyunsaturated fatty
acids (Maia et al 2010).
LACTATE-DEGRADING BACTERIA
Lactate is an intermediary product of ruminal fermentation, which is metabolized to VFAs. In diets rich in
starch, the population of this type of bacteria capable of
using lactic acid is increased (Counotte and Prins 1981).
Megasphaera elsdenii is the main species responsible
for lactic acid metabolization; thus, it has an important
role in the prevention of acidosis during the adaptation
period when ruminants are fed diets high in concentrate
(Counotte et al 1981).
PROTOZOA IN THE RUMEN
Protozoa constitute 40-80% of the biomass, most abundant (table 2) of which are the orders Entodiniomorphida
and Holotricha (Firkins et al 2007, Yáñez-Ruiz et al 2004).
The flow of ruminal protozoa to the ruminant abomasum
is less than that of bacteria, since they are retained in the
feed particles (Hook et al 2012). Holotrichs can assimilate
soluble sugars and keep some of them in reserve polysaccharides; thus, this protozoa can decrease the risk of acidosis
after consuming foods with high concentrations of easily
digestible sugars (van Zwieten et al 2008).
CELLULOLYTIC PROTOZOA
Approximately 90% of total protozoa belong to the
genus Entodiniomorphida, many of which are involved in
the hydrolysis and fermentation of cellulose (Yáñez-Ruiz
et al 2004). In in vitro studies with cultured protozoa, it was
observed that crystalline cellulose is degraded mainly by
protozoa of the genera Polyplastron and Eudiplodinium and
to a lesser degree by Epidinium (Fondevila and Dehority
2001). Besides having the ability to digest cellulose,
Table 2. Main ruminal protoza and fungi.
Principales protozoarios y hongos ruminales.
Protozoa
Cellullolytic protozoa
Enoploplastron triloricatum
Eudiplodinium maggii
Diploplastron affine
Epidinium ecaudatum
Diplodinium monacanthum
Diplodinium pentacanthum
Proteolytic protozoa
Entodinium caudatum
Eudiplodinium medium
Fungi
Cullulolytic fungi
Neocallimastix frontalis
Piromyces communis
Orpinomyces joyonii
Fermentation products
Reference
Reducing sugars
(Coleman et al 1976)
Amonium, VFA
Amonium, VFA
(Ivan et al 2000)
(Forsberg et al 1984)
Fermentation products
Reference
Lactate, Formate, Acetate, Succinate, Ethanol
Celobiose, celooligosacarides,
Glucose
(Moniello et al 1996)
(Dashtban et al 2009)
(Hodrova et al 1995)
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CASTILLO-GONZÁLEZ ET AL
Diploplastron affine has amylolytic activity; due to its
ability to produce amylolytic enzymes, including two
isoforms of α-amylase and maltase, it produces maltose,
maltotriose, and glucose (Wereszka and Michalowski
2012). Proteolytic protozoa: In the ruminal environment,
soluble proteins are mostly degraded by bacteria and protozoa (Hino and Russell 1987). The proteolytic activity
of ruminal bacteria is 6 to 10 times greater than that of
protozoa (Brock et al 1982).
RUMINAL FUNGI
Fungi (table 2) represent a small proportion, approximately 8%, of the biomass in the ruminal ecosystem
(Jenkins et al 2008), but they do have a role in the digestion
of food consumed by the ruminant (Nam and Garnsworthy
2007). Some fungi are microaerotolerants and are attached
to feed particles through a system of rhizoids (Denman
et al 2008). Ruminal fungal populations are favored by the
consumption of fibrous forage that is mainly highly lignified. Ruminal fungi are present in the duodenum, cecum,
and feces and are removed when ruminants are fed high
concentrations of rapidly fermentable sugars; however,
the fungi quickly proliferate once the feed concentration
is increased (Grenet et al 1989).
CELLULOLYTIC FUNGI
Ruminal fungi (table 2) are able to produce enzymes that
hydrolyze cellulose and xylans. Their enzymatic activity
is variable, depending on their phylogenetic origin and
especially their rhizoidal structure, but it has been postulated that some species, such as Neocallimastix frontalis,
Piromyces joyonii, and Orpinomyces communis, can more
efficiently digest structural polysaccharides than cellulolytic
bacterial species in monoculture (Bernalier et al 1992). The
fungal activity helps the ruminal digestion of the plant cell
wall. The production of zoospores by chemotaxis allows
rapid adhesion to the particles, then the fungi fracture
zones of lignified tissues by mechanical action, and the
nonlignified plant tissues are rapidly degraded (Grenet
et al 1989). Thus, ruminal fungi are particularly important
when the ruminant consumes many lignified substrates.
For example, N. frontalis has the ability to solubilize small
lignin fractions of the plant cell wall, allowing the bacteria
access to the cellulose (Borneman et al 1991).
MANIPULATION OF RUMINAL FERMENTATION
Biotechnology has been used to modify ruminal fermentation by promoting or diminishing certain fermentation
processes, leading to a higher efficiency in animal productivity. This biotechnological management can be categorized
into the following five groups: 1) modification of diet and
fermentation profile, 2) transformation of food before consumption, 3) manipulation of ruminal microorganisms, 4)
354
use of microorganism fermentation activators, and 5) use
of substances with ruminal activity.
MANAGEMENT OF DIET AND MODIFICATION
OF FERMENTATION PROFILE
Some diet changes may improve the microbial fermentation profile. For example, the inclusion of legumes
to the ruminant diet can have positive results in terms of
microbial activity and fermentation end products. Legumes
are a good source of protein; rich in amino acids, vitamins,
and minerals; and good substrates of cellulolytic microorganisms for growth and enzyme function (Galindo and
Marrero 2005). On the other hand, forages of low nutritional
value exist which contain high amounts of lignincellulose,
low concentrations of fermentable sugars, and proteins
of low quality; these characteristics affect the microbial
activity and thus the profile of the fermentation products.
For example, the use of rice straw decreases cellulolytic,
proteolytic, and amylolytic bacterial populations and the
total protozoa population; if these agricultural byproducts
are treated with urea, the microorganism can use it as a
substrate, leading to an increase of these populations and
improvement of the fermentation profile (Wanapat 2000).
FOOD TRANSFORMATION BEFORE
CONSUMPTION
Food transformation before consumption involves
all of the physical and chemical treatments applied to
food before being consumed by the ruminant. Physical
treatments include heating (Brandt and Klopfenstein
1986) and varying the particle sizes (Schadt et al 2012).
On the other hand, chemical treatments are the best used
methods, including the use of enzymes such as cellulases
and xylanases (Giraldo et al 2007). The ruminant diet is
based on forage consumption, in ruminant production systems, anything that improves the nutritional value of forage
with a high fiber concentration and low digestibility can
increase the productivity of ruminants (Giraldo et al 2007).
Researchers have shown that supplementing the cattle diet
with fibrolytic enzymes significantly improves the use of
nutrients and increases ruminant efficiency. The primary
enzymes used for this objective are xylanases (Goncalves
et al 2012) and cellulase (Morgavi et al 2001), which are
purified from fermentation cultures of both bacteria and
fungi (Beauchemin et al 2003). Several of these fibrolytic
enzymes have been evaluated as additives in ruminant
diets and were originally developed as additives in silage,
hay, and agricultural byproducts (Tang et al 2008). For
example, Tang et al (2008), proved that fibrolytic enzymes
supplementation increased in vitro digestibility of dry
matter and in vitro organic matter digestibility of maize
stover, maize stover silage, rice straw and wheat straw.
The exogenous enzymes that improve fibrolytic activity
favor the access of ruminal microbes to the cell wall
ADDITIVES, RUMINAL MICROORGANISMS, SYMBIOSIS
matrix, thus favoring fiber digestion (Nsereko et al 2000).
Cellulose and hemicellulose are the main polysaccharides
that form part of the cell wall (Hindrichsen et al 2006).
These polysaccharides are insoluble; but in the presence
of enzymes, such as amylases, proteases, and pectinases,
they are converted to soluble sugars so that they can be
used by ruminal microorganisms. Furthermore, these
enzymes may have secondary activities (Beauchemin
et al 2003). The improvement in ruminant efficiency due
to the use of fibrolytic enzymes is attributed to improved
ruminal forage digestion, which results in an increase in
dry matter digestibility and voluntary intake, increasing the
digestible energy that is used by the ruminant (Beauchemin
et al 2003, Krueger et al 2008). Also cellulase has been
shown to increase protein degradation of forages in vitro,
because proteins are more available to proteolytic enzymes
(Kohn and Allen 1992). Since intake of fibrolytic enzymes
improve the forage digestibility, it increases the total digestible nutrients and consequently changes concentration
and proportion of VFA, therefore, ruminant productivity
improves (Pinos-Rodríguez et al 2002, Pinos-Rodríguez
et al 2008). The enzyme products with this activity used
in ruminant nutrition are usually produced by fungi such
as Trichoderma longibrachiantum, Aspergillus niger,
Penicillium funiculosum (Wallace et al 2001) and A. oryzae,
as well as Bacillus spp. bacteria (Beauchemin et al 2003).
MANIPULATION OF RUMINAL
MICROORGANISMS
Despite the importance of ruminal microorganisms,
protozoa are not vital but are important for these animals.
It has been shown that protozoa can be eliminated from the
ruminal environment, defaunation causes changes in the
characteristics of ruminal digestion, such as an increase in
the bacterial density, degradation of starch, and decreasing
propionate and methane concentrations; in addition, fiber
digestion is affected (Morgavi et al 2010). Because protozoa are H+ producing microorganisms, this ion is used by
methanogens to reduce CO2 to CH4, it is considered that
the removal of protozoa decreases methanogenesis due to
the reduction of available H+ for methanogens (Mosoni
et al 2011). Moreover, defaunation causes changes in the
production of ammonia and the VFA profile (Ozutsumi
et al 2005). The amount of ammonia is less in defaunated
animals due to decreased proteolysis (Firkins et al 2007).
Therefore, although protozoa are not essential for ruminant
growth, its presence is important because of its ability to
degrade the main components, which is an important role
in the fermentation process (Coleman 1985). In addition,
the decrease in postprandial pH is regulated by protozoa
(Belanche et al 2011) because they modulate amylolytic
bacterial populations, which use glucose from starch as a
substrate for its fermentation processes to give lactate as one
of its main products (Mendoza et al 1993). Belanche et al
(2011), showed that presence of rumen protozoa increased
rumen total VFA and ammonia-N significantly, when the
ruminal protozoa are present. Hence there exists a positive
response on fiber digestibility and VFA concentration
to fautation, especially when structural carbohydrates
represent the principal dietary component, this confirms
the actions of protozoa to fribrolytic activity (Belanche
et al 2011). Nevertheless, it remains unclear whether this
increment in fiber digestibility is attributed specifically to
the protozoal activity, or to synergy between all ruminal
microorganisms (Chaudhary et al 1995).
USE OF MICROORGANISM FERMENTATION
ACTIVATORS
Probiotics are microorganisms that are not of ruminal origin but can be adapted to ruminal conditions and
improve the ruminal fermentation process. Probiotics
are defined as live microorganisms or components of
microbial cells that have beneficial effects on the host,
since they regulate the intestinal flora to improve animal
health, these products are named “direct-fed microbial”
instead of probiotic (Krehbiel et al 2003). The probiotics
used in ruminant feed mainly include fungi and bacteria,
which have replaced conventional antibiotics as growth
promoters (Lila et al 2004). Specific strains of bacteria
are used for this purpose in animal feed (Donohue 2006).
In beef cattle production systems, antibiotics are used to
decrease the frequency of metabolic disorders, improve
efficiency, and reduce ruminal acidosis, however, the use
of some unnatural antibiotics is restricted (Zerby et al
2011). Some bacterial species of the genera Lactobacillus
and Bifidobacterium have been shown to have good results
as probiotics in ruminants, increasing levels of weight
gain and efficiency (Whitley et al 2009). Furthermore,
it has been seen that these lactic acid bacteria are able to
reduce the risk of enteric disease caused by E. coli and
Salmonella spp. (Stephens et al 2007). The fungi Aspergillus
oryzae, Saccharomyces cerevisiae (Beharka et al 1991),
and Candida levica 25 (Marrero et al 2011) have been
used as probiotics as well as food additives to improve
the ruminal fermentation process. A. oryzae improves the
use of lactate by several bacteria such as Megasphaera
elsdenii and Selomonas ruminantium (Beharka et al 1991),
thus avoiding the drop in pH immediately after eating
(Moya et al 2009). S. cerevisiae favors the metabolism of
ruminal microorganisms (Oeztuerk et al 2005) because it
increases the ruminal pH or decreases the time in which
the pH is less than 5.6 (Thrune et al 2009), increases
the ratio of butyrate and propionate (Pinos-Rodríguez
et al 2008), and increases the digestibility of dry matter
and neutral detergent fiber (Lila et al 2004). Also, yeast
cultures stimulate the use of H+ by acetogenic bacteria,
thereby decreasing methane production (Chaucheyras et al
1995). The presence of Saccharomyces cerevisiae favors
the establishment of fibrolytic bacteria as F. succinogenes
and R. albus (Callaway and Martin 1997), and decreases
355
CASTILLO-GONZÁLEZ ET AL
the population of lactate-producing microorganisms and
those using H+ to produce CH4 (Lila et al 2004). In addition, S. cerevisiae stimulates propagation of the fungus N.
frontalis and lactate production using M. elsdenii and S.
ruminantium (Chaucheyras et al 1995), preventing ruminal
pH decreases (Lynch and Martin 2002), improving fiber
fermentation, and increasing propionate production (Lila
et al 2004). All of these results suggest that these additives
are beneficial in terms of weight gain (Tricarico et al 2007)
and milk production (Dann et al 2005). Furthermore, DFM
decreased fecal excretion of E.coli O157:H7 from infected
calves, therefore a possible application for DFM might be
to reduce shedding of this pathogen from cattle (Krehbiel
et al 2003). In ruminants, probiotics have shown positive
effects in cattle at weaning and increased production of
dairy cows, nutrient absorption, efficiency, weight gain in
beef cattle as well as greater resistance to gastrointestinal
diseases (Krehbiel et al 2003).
USE OF SUBSTANCES WITH RUMINAL ACTIVITY
The use of substances with ruminal activity refers
to the use of additives or supplements added to animals
with the objective to potentiate the fermentation processes
taking place in the rumen, thus, greater efficiency in the
utilization of food is achieved.
NITROGENOUS SUPPLEMENTS
Feeding ruminants with agricultural byproducts is
an economical option to cover the energy requirements
from fiber and maintenance of the animal; however, the
crude protein content is not adequate, which requires
supplementation with nitrogenous compounds (Farmer
et al 2004). The use of non-protein nitrogen such as urea
is a good strategy to cover this need (Currier et al 2004),
but its benefit in efficiency is lower when the animal is
supplemented with low quality forage with natural sources of protein. Furthermore, supplementation with crude
protein (CP) to ruminants improves fiber digestibility
because the population of cellulolytic bacteria is increased
(Currier et al 2004). Schauver et al (2005) suggest that
providing a protein supplement daily to cows grazing low
quality forage increases body weight (BW) and decreases
the grazing time. Also, there exists a synergy between
protein and sugar supplementation, because the microbial
CP synthesis is improved to a greater extent when protein
is supplied in addition to sugar rather that when they are
provided separately.
USE OF PH BUFFERING SUBSTANCES
Increased propionate levels at the expense of acetate
are generally associated with high concentrate fattening
rations. Excessive lactic acid accumulations can occur on
high concentrate diets and produce a pH drop. The use of
356
pH buffering compounds has the ability to maintain the
ruminal pH necessary to provide good microbial activity.
For this purpose, mineral salts such as carbonates, bicarbonates, and phosphates of sodium, potassium, calcium,
and magnesium are used. These compounds maintain a
constant pH and promote the population of cellulolytic
bacteria, thereby increasing the digestibility of dry matter
and protein synthesis (Galindo and Marrero 2005) and
preventing acidosis.
IONOPHORES
Chemically, ionophores are polyether carboxylic antibiotics, which initially were used as anticoccidial agents in
poultry. These compounds are produced by various strains of
Streptomyces and include moensin, lasalocid, salinomycin,
and narasin (Bergen and Bates 1984). These compounds
are used as growth promoters (Bretschneider et al 2008).
In the 1970s, the US Food and Drug Administration approved the use of the ionophore monensin as an additive
in ruminant diets. However, as the use of antibiotics in
animal feed may leave residues of these compounds in
animal products allowing the development of pathogens
resistant to antibiotics, Europe has prohibited the use of
sodium monensin since January of 2006 (Zawadzki et al
2011). Monensin is an antibiotic produced by the fungus
Streptomyces cinnamonensis (Li et al 2009), which acts
mainly by affecting Gram-positive bacteria. Monensin
binds to the cell membrane of these bacteria, causing monovalent proton ion exchange, a decrease in intracellular
K+ and Na+ accumulation, and a loss of cellular energy
(Ellis et al 2012). Ruminal acidosis is one of the main and
most common metabolic disorders in cattle. This disorder
is caused by a diet with a high proportion of concentrate,
which is currently used in animal production and is related
to lactic acid production, but excessive production of VFA
may be a more important contributor to chronic acidosis
problems in dairy cows, because it causes a pH below
5.5 (McGuffey et al 2001). Ionophores also have been
used in ruminants due to their ability to reduce the risk of
bloating, increase the efficiency in the animal (Bergen and
Bates 1984, Bretschneider 2010), reduce the production of
methane (gas trapped in bubbles which produce the bloat)
by controlling the bacteria that produce H+ used in dioxide
reduction and acetate (Bretschneider 2010), increase the
production of propionate, and improve the acetate:propionate
ratio (Russell and Mantovani 2002), thereby increasing the
holding energy (Marrero et al 2011). In ruminants that are
fed high concentrate diets, ionophores usually reduce feed
intake and improve animal weight gain. On the other hand,
when the ruminant diet contains significant concentrations
of cellulose, as in the case of forage, ionophores do not
reduce consumption and improve weight gain, improving
feed conversion in both cases (Bergen and Bates 1984).
Monensin is one of the most used ionophores in ruminant
nutrition. The main benefit observed with the use of this
ADDITIVES, RUMINAL MICROORGANISMS, SYMBIOSIS
ionophore is the increase in the molar proportion of propionic acid at the expense of a decrease in the acetic acid and
butyric acid concentrations produced in the rumen. These
changes in the profile of VFAs are related to a decrease in
methane concentration (Bergen and Bates 1984).
ciliated protozoa populations, however, yellow grease
supplementation did not affect numbers of protozoa in
steers fed either sorghum or corn diets (Towne et al 1990).
TALLOW
Ruminal fermentation is the result of metabolism of
bacteria, fungi, and protozoa that are present in this environment. Their metabolic pathways are interwoven so that
the end product or intermediate metabolites of any type of
microorganism is the substrate of another, thus achieving
the end products of ruminal fermentation necessary for
ruminant nutrition. This situation creates interdependence
between different microorganisms, and these emerging
consortia facilitate their establishment. Modern meat and
milk productions tend to use diets that can be challenging
for ruminants, or rather for ruminal microorganisms. The
microorganisms’ responses to these challenges are not
always beneficial to the animal. So, experts have speculated on ways to improve ruminal fermentation to increase
animal production. The use of additives can manipulate
the ruminal ecosystem and ruminal microflora in order to
improve production. Because ruminal microorganisms are
crucial for proper animal nutrition, it is important to generate
innovative knowledge in the study of ruminal fermentation
and microbial ecosystems to improve the ruminant feeding
process. Today, molecular tools are being developed to
better understand the symbiosis between microorganisms
and ruminants. In addition, the environmental impact due
to cattle production is a current concern. Therefore, the
use of additives in the diet to improve the efficient use of
nutrients and thus reduce the final products that affect the
atmosphere are being developed. Prospects indicate that the
use of combinations of additives can be added to ruminant
rations. These additives will improve animal production
with minimal possible damage to the environment. Thus,
molecular techniques will identify microbial population
Holotrichos changes during fermentation so that it may
be possible to manipulate and stimulate the presence of
microorganism populations capable of providing a better
nutritional benefit to the animal.
It is an abundant and low cost source of supplemental
fat used to increased dietary energy density for dairy cows
(Ruppert et al 2003).The objective of tallow supplementation in the diets of dairy and beef cattle is to increase the
energy density to improve milk production and metabolic
efficiency of cattle (Grummer and Carroll 1991). It has
been demonstrated that native tallow did not negatively
affect ruminal fermentation, nutrient digestibility, dry
matter intake, or milk production when supplemented
in typically recommended amounts (e.g., 2 to 3% of dry
matter) to high producing dairy cows when the basal diet
consisting of alfalfa hay or silage as the principal forage
(Ruppert et al 2003), so the dairy cattle industry routinely adds fat to promote milk production (Appeddu et al
2004). On the other hand, one experiment showed that
supplementation of either tallow or choice white grease to
a diet in which corn silage was the only forage decreased
dry matter intake, milk yield, milk fat percentage, and the
ruminal acetate to propionate ratio, but neither fat source
affected in situ dry matter intake or neutral detergent fiber
disappearance (Onetti et al 2001). Another important effect
of fat supplementation is an improved reproductive development and reduced risk of metabolic disorders, due to
the removal of fat from body stores (Dann et al 2005). In
addition, inadequate energy intake during the prepartum
and early lactation periods is associated with metabolic
disorders such as ketosis (Dann et al 2005), fatty liver,
and low reproductive response (Douglas et al 2007). If
high producer cows are supplemented with fat, energy
consumption is increased and the effect of a negative
energy balance is reduced, thereby improving the health
of the dairy cows (Grummer and Carroll 1991). During
lactation, feed high in available protein, starch, and /or
sugar are commonly supplemented to promote pre weaning
gains in offspring and to prevent excessive BW losses of
grazing cows and ewes. Casals et al (1999) reported that
4 wk old lambs increased their weights when supplying
fat to ewes consuming a high forage diet. However, an
excess (> 3-5%) of tallow in the diet has been associated
with a decrease in the palatability and digestibility of fiber.
These negative effects occur due to a toxic effect of the
long chain fatty acids on ruminal microorganisms, mainly
bacteria (Henderson 1973). On the other hand, when the
amount is not exceeded, a small increase of fat may improve
bacterial growth by incorporating the dietary fatty acids
and reducing the need for synthesizing them (Amorocho
et al 2009). There also have been reports that fattening
steers with tallow supplementation causes a decrease of
CONCLUSION
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